An inability to arrest at cell cycle checkpoints is a hallmark of cancer cells that affects their response to chemotherapeutic drugs. In response to DNA damage normal cells arrest in both G1 and G2. The ability to arrest in G1 is mediated by p53, and this tumor suppressor is mutated or lost in the majority of tumors. P53 activates the transcription of p21, a CDK inhibitor that blocks the activity of G1 cyclin/CDK complexes. Although a role for p53 in the G2 checkpoint has been suggested, direct evidence implicating p53 in control of G2 or its cyclin/CDK complex has not been demonstrated. G2 arrest mediated by inhibition of cyclin B/CDC2 activity is regulated by CDC2 phosphorylation and/or cyclin B levels. Our data, using a model system in which p53 is depleted from otherwise normal cells by transduction with an HPV 16 E6-expressing retrovirus has provided new insights into regulation of the G2 checkpoint by p53. We hypothesize that the G2/M transition is regulated by redundant G2 checkpoints; in cells with functional p53, G2 arrest in response to DNA damage is mediated by down-regulation of cyclin B and CDC2 transcripts and the consequent inactivation of CDC2/cyclin B kinase, whereas in cells lacking p53, G2 arrest occurs despite active cyclin B/CDC2 kinase. We propose to identify the redundant DNA damage induced G2 arrest pathway that assures arrest in G2 independently of CDC2 activity. We propose to determine what role p53 plays in the regulation of cyclin GB and CDC2 transcription by promoter-reporter assays performed on cells depleted of p53 HPV E6, or depleted of p21 by homologous recombination. Additionally, we have found that cells lacking p53 (but not cells with intact p53) lose their DNA damage induced G2 checkpoint over their proliferative life span. We propose to investigate the mechanism by which this attenuation occurs. Finally, we propose to use our model system to determine how loss of p53, and the G2 checkpoint, affects the response to chemotherapeutic drugs, and to identify additional genes that mediate the chemoresistance frequently observed clinically in p53 mutated tumors.